Processes directed towards immobilising proteins on to a solid surface are of considerable commercial interest. Functionalisation of surfaces with immunological materials, such as antibodies or antibody fragments, for example, forms the basis of immunoadsorption techniques such as immuno affinity purification processes which are increasingly being applied to the recovery or purification of a range of commercially important materials.
Commonly, attachment of proteins to solid surfaces, such as chromatography media, has been brought about by exposing the surface to a solution of the protein such that the protein is adsorbed onto the solid surface via non-specific binding mechanisms. Methods for immobilising proteins on chromatography media are well established in the literature (see for example, In Protein Immobilisation, R. F. Taylor ed., Marcel Dekker, Inc., New York, 1991). Where the solid surface is provided by a hydrophobic material such as polystyrene, for example, then attachment is generally brought about by adsorption of hydrophobic regions of the protein onto the hydrophobic surface.
Adsorption onto the solid surface is usually accompanied by significant conformational disruption with partial unfolding and denaturation of the protein concerned. The concomitant loss of protein activity detracts from the overall usefulness of the process. Commonly, for example, adsorption of antibodies onto a hydrophobic surface is accompanied by the loss of in the order of greater than 95% of specific binding activity. Where smaller antibody fragments are involved, the amount of specific binding affinity retained upon adsorption onto a solid surface can be even lower as described in Molina-Bolivar et al, J. Biomaterials Science-Polymer Edition, 9, 1103-1113, 1998).
Alternatives to or improvements upon the method of adsorption of proteins in the preparation of immobilised protein surfaces have been considered.
One alternative approach is to use chemical cross-linking of residues in the protein for covalent attachment to an activated solid surface using conventional coupling chemistries for example as described in Bioconjugate Techniques, G. T. Hermanson, ed. Academic Press, Inc., San Diego, Calif., USA. Amino acid residues incorporating sulphydryl groups, such as cysteine, may be covalently attached using a bispecific reagent such as succinimidyl-maleimidophenylbutyrate (SMPB), for example. Alternatively, lysine groups located at the protein surface may be coupled to activated carboxyl groups at the solid surface by conventional carbodiimide coupling using 1, ethyl-3-[3-dimethyl aminopropyl]carbodiimide (EDC) and N-hydroxysuccinimide (NHS). A disadvantage of this approach is that cross-linking residues in the protein can interfere with the functionality of the protein.
By providing the protein with a peptide tail extension containing cross-linkable residues, coupling of the protein to the surface can be brought about using conventional chemical cross-linking agents at a site remote from the main body of the protein. In this way, the covalent coupling process itself is less likely to interfere with the functionality of the protein.
EP 0434317 (Joseph Crosfield & Sons) discloses the use of improved affinity purification media which employ small specific binding agents, especially Fv antibody fragments. These optionally have a hydrophobic tail, with a particularly preferred linking group being the residue “Myc” amino acid sequence. Although such a group is primarily intended to facilitate immobilisation of the binding agent by non-covalent attachment onto a hydrophobic surface, it is mentioned in passing in the specification that as the myc group contains a lysine residue, it could also be used for covalent attachment onto surfaces.
Alternative peptide tails incorporating histidine residues have been used to attach proteins to nitrilotriacetic acid (NTA, manufactured by QIAGEN GmbH surfaces through the co-ordination of nickel. However, interactions of this type are non-covalent.
There remains a continuing need to improve the efficiency of the coupling reaction, however, and in particular to address the problems arising from the need to ensure that the protein is brought into association with the surface prior to the covalent coupling reaction. In case of coupling a protein to a negatively charged surface, such as a carboxymethyl activated dextran surface, for example, the coupling reaction must be performed at a low pH to ensure that the protein is positively charged in order for such association to occur. As many proteins are acid-sensitive, such conditions may have the effect of impairing the specific activity of the cross-linked protein. Not only would it be desirable to increase the amount of protein coupled to the surface but also to increase the proportion of coupled protein which retains its specific activity by minimising the possibility of non-specific binding interactions resulting from the protein unfolding under the coupling conditions.